RESUMEN
The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.
Asunto(s)
Imagenología Tridimensional , Riñón , Humanos , Riñón/diagnóstico por imagen , Imagenología Tridimensional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Aprendizaje AutomáticoRESUMEN
Studies of female genital structures have generally lagged behind comparable studies of male genitalia, in part because of an assumption of a lower level of variability, but also because internal genitalia are much more difficult to study. Using multiple microscopy techniques, including video stereomicroscopy, fluorescence microscopy, low-temperature scanning electron microscopy (LT-SEM), and confocal laser scanning microscopy (CLSM) we examined whether the complex sperm transfer structures in males of Megalolaelaps colossus (Acari: Mesostigmata) are matched by similarly complex internal structures in the female. While both LT-SEM and CLSM are well suited for obtaining high-quality surface images, CLSM also proved to be a valuable technique for observing internal anatomical structures. The long and coiled sperm transfer organ on the chelicera of the males (spermatodactyl) largely matches an equally complex, but internal, spiral structure in the females in shape, size, and direction. This result strongly suggests some form of genital coevolution. A hypothesis of sexual conflict appears to provide the best fit for all available data (morphology and life history).
Asunto(s)
Genitales Femeninos/anatomía & histología , Genitales Masculinos/anatomía & histología , Imagenología Tridimensional , Microscopía Confocal , Ácaros/anatomía & histología , Animales , Femenino , Genitales Femeninos/ultraestructura , Genitales Masculinos/ultraestructura , Masculino , Tamaño de los Órganos , Reproducción/fisiologíaRESUMEN
Single-cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single-nuclear sequencing data sets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell-type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of 2 murine AKI models: ischemia/reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single-cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature, and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by indEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complemented single-cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.
Asunto(s)
Lesión Renal Aguda , Células Epiteliales , Transcriptoma , Lesión Renal Aguda/inmunología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Humanos , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Ratones , Persona de Mediana Edad , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Análisis de la Célula Individual , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
Ostertagia ostertagi is an abomasal parasite with significant economic impact on the cattle industry. Early host immune responses are poorly understood. Here, we examined time course expression of Toll-like receptors (TLRs) in peripheral blood mononuclear cells (PBMC) during infection where PBMC macrophages (MÏ) generated both pro- and anti-inflammatory responses when incubated with excretory/secretory products (ESP) from fourth-stage larvae (OoESP-L4) or adult worms (OoESP-Ad). First, changes in cell morphology clearly showed that both OoESP-L4 and OoESP-Ad activated PBMC-MÏ in vitro, resulting in suppressed CD40 and increased CD80 expression. Expression of mRNAs for TLR1, -4, -5, and -7 peaked 7 days postinfection (dpi) (early L4), decreased by 19 dpi (postemergent L4 and adults) and then increased at 27 dpi (late adults). The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) (transcript and protein) increased in the presence of OoESP-Ad, and the anti-inflammatory cytokine interleukin 10 (IL-10) (protein) decreased in the presence of OoESP-L4 or OoESP-Ad; however, IL-10 mRNA was upregulated, and IL-6 (protein) was downregulated by OoESP-L4. When PBMC-MÏ were treated with ligands for TLR4 or TLR5 in combination with OoESP-Ad, the transcripts for TNF-α, IL-1, IL-6, and IL-10 were significantly downregulated relative to treatment with TLR4 and TLR5 ligands only. However, the effects of TLR2 ligand and OoESP-Ad were additive, but only at the lower concentration. We propose that O. ostertagi L4 and adult worms utilize competing strategies via TLRs and MÏ to confuse the immune system, which allows the worm to evade the host innate responses.
Asunto(s)
Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ostertagia/inmunología , Ostertagiasis/veterinaria , Receptores Toll-Like/metabolismo , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Citocinas/metabolismo , Interacciones Huésped-Parásitos/inmunología , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Transducción de SeñalRESUMEN
Tetranychus canadensis (McGregor) is redescribed based on type specimens and American non-type specimens. The ontogenetic development of leg chaetotaxy is provided, which is the typical additional pattern for Tetranychus. The variation of pregenital striae and shape of aedeagi are discussed. Low temperature scanning electron microscopic photos show the supracoxal setae on palpfemur (ep) and leg coxae I (el) are eupathidia; lateral and ventral lips bear modified leaf-like adoral setae (or2 and or3) and the dorsal lips bearing a pair of spine-like dorsal adoral setae (or1); dorsal seta on tibia I (db) is a trichobothrium with a cup-shaped base and broken striae on the inner integument. Confocal scanning shows a thin, long ejaculatory duct and a thicker duct connecting the aedeagus to a cup-shaped seminal vesicle; the aedeagus is hollow where the ejaculatory duct passes through.
Asunto(s)
Tetranychidae , Animales , Frío , Integumento Común , Microscopía Electrónica de Rastreo , PielRESUMEN
Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species.IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.
Asunto(s)
Campylobacter jejuni/fisiología , Flagelos/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Evolución Biológica , Campylobacter jejuni/clasificación , Relación Estructura-Actividad , Sistemas de Secreción Tipo IIIRESUMEN
Feeding behaviors and biomechanics of female Varroa destructor mites are revealed from AC-DC electropenetrography (EPG) recordings of mites feeding from Apis mellifera honey bee pupae and histology of mite internal ingestion apparatus. EPG signals characteristic of arthropod suction feeding (ingestion) were identified for mites that fed on pupae during overnight recordings. Ingestion by these mites was confirmed afterwards by observing internally fluorescent microbeads previously injected into their hosts. Micrographs of internal ingestion apparatus illustrate the connection between a gnathosomal tube and a pharyngeal lumen, which is surrounded by alternating dilator and constrictor muscles. Inspection of EPG signals showed the muscularized mite pharyngeal pump operates at a mean repetition rate of 4.5â¯cycles/s to ingest host fluids. Separate feeding events observed for mites numbered between 23 and 33 over approximately 16â¯h of recording, with each event lasting ~10â¯s. Feeding events were each separated by ~2â¯min. Consecutive feeding events separated by either locomotion or prolonged periods of quiescence were grouped into feeding bouts, which ranged in number from one to six. Statistical analyses of EPG data revealed that feeding events were prolonged for mites having lower pharyngeal pump frequencies, and mites having prolonged feeding events went unfed for significantly more time between feeding events. These results suggest that mites may adjust behaviors to meet limitations of their feeding apparatus to acquire similar amounts of food. Data reported here help to provide a more robust view of Varroa mite feeding than those previously reported and are both reminiscent of, as well as distinct from, some other acarines and fluid-feeding insects.
Asunto(s)
Abejas/parasitología , Conducta Alimentaria/fisiología , Varroidae/fisiología , Animales , Fenómenos Biomecánicos , Fenómenos Electrofisiológicos , Femenino , Microesferas , Faringe/inervación , Faringe/fisiología , Pupa/parasitologíaRESUMEN
The parasitic mite Varroa destructor is the greatest single driver of the global honey bee health decline. Better understanding of the association of this parasite and its host is critical to developing sustainable management practices. Our work shows that this parasite is not consuming hemolymph, as has been the accepted view, but damages host bees by consuming fat body, a tissue roughly analogous to the mammalian liver. Both hemolymph and fat body in honey bees were marked with fluorescent biostains. The fluorescence profile in the guts of mites allowed to feed on these bees was very different from that of the hemolymph of the host bee but consistently matched the fluorescence profile unique to the fat body. Via transmission electron microscopy, we observed externally digested fat body tissue in the wounds of parasitized bees. Mites in their reproductive phase were then fed a diet composed of one or both tissues. Mites fed hemolymph showed fitness metrics no different from the starved control. Mites fed fat body survived longer and produced more eggs than those fed hemolymph, suggesting that fat body is integral to their diet when feeding on brood as well. Collectively, these findings strongly suggest that Varroa are exploiting the fat body as their primary source of sustenance: a tissue integral to proper immune function, pesticide detoxification, overwinter survival, and several other essential processes in healthy bees. These findings underscore a need to revisit our understanding of this parasite and its impacts, both direct and indirect, on honey bee health.
Asunto(s)
Abejas/parasitología , Cuerpo Adiposo/parasitología , Hemolinfa/parasitología , Varroidae/patogenicidad , Animales , Dieta , Interacciones Huésped-Parásitos/fisiología , Reproducción/fisiologíaRESUMEN
The genus Ceratotarsonemus De Leon (Acari: Prostigmata: Tarsonemidae) is reviewed here, with the addition of an updated key for the genus. Ceratotarsonemus amazonicus, sp. nov., found in the Brazilian Amazon rainforest, is described. Phase contrast (PC), differential interference contrast (DIC), low temperature scanning electron microscopy (LT-SEM) and confocal microscopy (CLSM) micrographs are provided. Biological and ecological aspects about the role of this species in its ecosystem are also discussed.
Asunto(s)
Ácaros y Garrapatas , Animales , Brasil , Ecosistema , Bosques , Bosque LluviosoRESUMEN
Campylobacter jejuni is an important human pathogen that causes 96 million cases of acute diarrheal disease worldwide each year. We have shown that C. jejuni CsrA is involved in the post-transcriptional regulation of more than 100 proteins, and altered expression of these proteins is presumably involved in the altered virulence-related phenotypes of a csrA mutant. Mutation of fliW results in C. jejuni cells that have greatly truncated flagella, are less motile, less able to form biofilms, and exhibit a reduced ability to colonize chicks. The loss of FliW results in the altered expression of 153 flagellar and non-flagellar proteins, the majority of which are members of the CsrA regulon. The number of proteins dysregulated in the fliW mutant was greater at mid-log phase (120 proteins) than at stationary phase (85 proteins); 52 proteins showed altered expression at both growth phases. Loss of FliW altered the growth-phase- and CsrA-mediated regulation of FlaA flagellin. FliW exerts these effects by binding to both FlaA and to CsrA, as evidenced by pull-down assays, protein-protein cross-linking, and size-exclusion chromatography. Taken together, these results show that CsrA-mediated regulation of both flagellar and non-flagellar proteins is modulated by direct binding of CsrA to the flagellar chaperone FliW. Changing FliW:CsrA stoichiometries at different growth phases allow C. jejuni to couple the expression of flagellar motility to metabolic and virulence characteristics.
Asunto(s)
Campylobacter jejuni/genética , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares/metabolismo , Regulón/genética , Proteínas Represoras/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Campylobacter jejuni/crecimiento & desarrollo , Pollos/microbiología , Flagelos/genética , Flagelina/genética , Flagelina/metabolismo , Chaperonas Moleculares/genética , Mutación , Unión Proteica , Proteómica , Proteínas Represoras/genéticaRESUMEN
Campylobacter jejuni infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, csrA mutant, and complemented csrA mutant C. jejuni strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the csrA mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The csrA mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5' end of flaA mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the csrA mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in C. jejuni colonization and pathogenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Campylobacter/metabolismo , Campylobacter jejuni/metabolismo , Campylobacter jejuni/patogenicidad , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Infecciones por Campylobacter/genética , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación Bacteriana de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Regulón/genética , Factores de Transcripción/genética , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuniâ FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuniâ FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuniâ FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output - precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility.